Sphingomyelin detecting probe

ABSTRACT

An object of the present invention is to provide a protein useful as a sphingomyelin detecting probe, which specifically recognizes sphingomyelin and has low cytotoxicity. The present invention provides a protein which has an amino acid sequence having, as the amino acid sequence from the 1st to the 48th amino acid, the amino acid sequence from the 1st to the 48th amino acid in Lysenin 1, and as the amino acid sequence from the 49th to the 298th amino acid, the amino acid sequence from the 51st to the 300th amino acid in Lysenin 3; and a protein which is obtained by deleting N terminal and/or C terminal from earthworm toxins Lysenin 1 or 3, and which specifically recognizes sphingomyelin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/138,634,filed May 6, 2002, which is incorporated by reference herein in itsentirety, and claims priority under 35 U.S.C. §119 of JapaneseApplication Nos. 2001-137087, filed May 8, 2001 and 2001-261158, filedAug. 30, 2001.

TECHNICAL FIELD

The present invention relates to novel proteins which are useful as asphingomyelin detecting probe. More particularly, the present inventionrelates to a chimera protein comprising Lysenin 1 and Lysenin 3 and aprotein of partial amino acid sequence of Lysenin 1 or Lysenin 3, bothof which specifically recognize sphingomyelin and have low cytotoxicity.The present invention relates to a gene which encodes said protein, avector having said gene, a transformant having said vector, a reagentfor detecting sphingomyelin comprising said protein, a method fordetecting sphingomyelin using said protein, and a kit for detectingsphingomyelin.

BACKGROUND TECHNIQUE

Major lipid components in cell membrane include glycerolipid,sphingolipid, and cholesterol. Glycerolipid in animal cells has astructure in which 2 molecules of a fatty acid and a phosphate group arebound to a glycerol skeleton, and has, upon dispersion in an aqueoussolution, a two layered membrane structure in which a hydrophobic fattyacid residue is present inside and a phosphate group portion is presentoutside. The formed two layered membrane structure has a property ofliquid crystalline, and in 1972 Singer and Nicolson (United States)proposed a “fluid mosaic model” in which a protein is floating in suchliquid crystal.

In addition to the cell membrane for separating the cell from outside,the cell also has a complicated membrane structure inside and hasorganelles having their own respective characteristic functions. Aportion which constitutes a cell membrane is synthesized by an organellereferred to as an “endoplasmic reticulum” and is finally transported toa cell membrane through a Golgi apparatus.

Glycerolipid which is one component of a cell membrane, contains a fattyacid and a phosphate group. However, the types of fatty acid arevarious, and the phosphate group portion also has variety, for example,it may contain choline, ethanolamine, serine, or inositol. Further,sphingolipid to which a variety of saccharides such as glucose,galactose, or lactose are bound, exists. There are several tens ofthousands of lipid molecules in the natural world if all the abovelipids are included. The composition of the lipid varies depending onthe type of organisms, the type of organs, the type of cells, and thetype of organelles. Furthermore, the lipid composition of the innerlayer constituting the lipid bilayer of the biomembrane is differentfrom that of the outer layer.

Unlike glycerolipid, since sphingolipid has a base referred to as“sphingosine” as a skeleton, it can be either donor or acceptor of ahydrogen bond. Also, in general, sphingolipid binds to a long-chainedfatty acid. Due to this structural character of sphingolipid, in thecase of a plurality of sphingolipids, the hydrophilic portions arelikely to aggregate through hydrogen bonding while the hydrophobicportions are likely to aggregate through the hydrophobic interaction offatty acid chains. Simons et al. (Germany) proposed that sphingolipidson the cell membrane aggregate and form a lipid domain. They named thisa “lipid raft” (hereinafter merely referred to as a “raft”).

Ever since the concept of the “raft” as described above was proposed, ithas been suggested that the raft plays an important role in the signaltransduction, as well as cell adhesion, infection of viruses andbacteria, polymer translocation in the cell, and the like. In recentyears, it has been shown that among sphingolipids, sphingomyelin is anecessary and enough factor in the formation of the raft.

The present inventors have heretofore found that Lysenin, which is atoxic protein secreted by Eisenia foetida, specifically binds tosphingomyelin.

Lysenin is already known and the existence of three types of Lysenin 1,2, and 3 is known. Lysenin 1 is composed of 297 amino acid residues, andLysenins 2 and 3 are composed of 300 amino acid residues. Among these,Lysenins 1 and 3 have high cytotoxicity and, thus, they could not beused for labeling sphingomyelin in living cells. Lysenin 2 does notrecognize sphingomyelin. Therefore, Lysenins which have been heretoforereported, could not be put to practical use as sphingomyelin detectingprobes.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide a proteinuseful as a sphingomyelin detecting probe, which specifically recognizessphingomyelin and has low cytotoxicity. Another object of the presentinvention is to provide a gene coding for the protein, a vector havingthe gene, a transformant having the vector, a reagent for detectingsphingomyelin which comprises the protein, a method for detectingsphingomyelin using the protein, and a kit for detecting sphingomyelin.

The present inventors have conducted concentrated studies in order toachieve the above objects, and as a result, they have succeeded inobtaining a protein which has low toxicity and specifically recognizessphingomyelin from Lysenin variants, thereby completing the presentinvention.

Thus, the present invention provides a protein having either of thefollowing amino acid sequences:

-   -   (1) an amino acid sequence having, as the amino acid sequence        from the 1st to the 48th amino acid, the amino acid sequence        from the 1st to the 48th amino acid in Lysenin 1, and as the        amino acid sequence from the 49th to the 298th amino acid, the        amino acid sequence from the 51st to the 300th amino acid in        Lysenin 3; or    -   (2) an amino acid sequence derived from the amino acid sequence        according to (1) above by substitution, deletion, and/or        addition of one or more amino acids, which specifically        recognizes sphingomyelin and has low cytotoxicity.

Another aspect of the present invention provides a protein having eitherof the following amino acid sequences:

-   -   (1) the amino acid sequence of SEQ ID NO: 3; or    -   (2) an amino acid sequence derived from the amino acid sequence        of SEQ ID NO: 3 by substitution, deletion, and/or addition of        one or more amino acids, which specifically recognizes        sphingomyelin and has low cytotoxicity.

A further aspect of the present invention provides a protein which isobtained by deleting N terminal and/or C terminal from earthworm toxinsLysenin 1 or 3, and which specifically recognizes sphingomyelin.Preferably, there is provided a protein obtained by deleting 50 to 200amino acid residues of N terminal and/or C terminal of earthworm toxinsLysenin 1 or 3.

A further aspect of the present invention provides a protein havingeither of the following amino acid sequences:

-   -   (1) the amino acid sequence of SEQ ID NO: 5; or    -   (2) an amino acid sequence derived from the amino acid sequence        of SEQ ID NO: 5 by substitution, deletion, and/or addition of        one or more amino acids, which specifically recognizes        sphingomyelin and has low cytotoxicity.

A further aspect of the present invention provides a gene which encodesthe above proteins of the present invention.

A still further aspect of the present invention provides a gene havingeither of the following nucleotide sequences:

-   -   (1) the nucleotide sequence of SEQ ID NO: 4; or    -   (2) a nucleotide sequence derived from the nucleotide sequence        of SEQ ID NO: 4 by substitution, deletion, and/or addition of        one or more nucleotides, which encodes a protein which        specifically recognizes sphingomyelin and has low cytotoxicity.

A still further aspect of the present invention provides a gene havingeither of the following nucleotide sequences:

-   -   (1) the nucleotide sequence of SEQ ID NO: 6; or    -   (2) a nucleotide sequence derived from the nucleotide sequence        of SEQ ID NO: 6 by substitution, deletion, and/or addition of        one or more nucleotides, which encodes a protein which        specifically recognizes sphingomyelin and has low cytotoxicity.

A still further aspect of the present invention provides a vector havingthe gene of the present invention.

A still further aspect of the present invention provides a transformanthaving the vector of the present invention.

A still further aspect of the present invention provides a reagent fordetecting sphingomyelin comprising the protein of the present invention.

A still further aspect of the present invention provides a method fordetecting sphingomyelin using the protein of the present invention.

A still further aspect of the present invention provides a kit fordetecting sphingomyelin, which comprises the protein, the gene, thevector, the transformant, or the reagent for detecting sphingomyelinaccording to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a result of an experiment for the confirmation ofreactivity between the protein of Example 1 of the present invention andsphingomyelin by ELISA (on left graph), and a result of hemolysisexperiment using the protein of Example 1 of the present (on rightgraph).

FIG. 2 shows a result of cell staining using the protein of Example 1 ofthe present invention and Niemann-Pick type A fibroblast.

FIG. 3 shows (1) a result of an experiment for the confirmation ofspecific reactivity between the protein of Example 1 of the presentinvention and sphingomyelin of ELISA, and (2) the chemical structures ofphosphatidylcholine and sphingomyelin.

FIG. 4 shows a result of an experiment for the confirmation ofreactivity between the protein of Example 2 of the present invention andsphingomyelin by ELISA.

FIG. 5 shows a result of hemolysis experiment using the protein ofExample 2 of the present.

FIG. 6 shows a result of cell staining using the protein of Example 2 ofthe present invention and Niemann-Pick type A fibroblast.

FIG. 7 shows a result of cell staining using the protein of Example 2 ofthe present invention and Niemann-Pick type A fibroblast.

DETAILED DESCRIPTION OF THE INVENTION

The embodiments of the present invention will now be described indetail.

(A) Proteins of the Present Invention

The first protein of the present invention has either of the followingamino acid sequences:

-   -   (1) an amino acid sequence having, as the amino acid sequence        from the 1st to the 48th amino acid, the amino acid sequence        from the 1st to the 48th amino acid in Lysenin 1, and as the        amino acid sequence from the 49th to the 298th amino acid, the        amino acid sequence from the 51st to the 300th amino acid in        Lysenin 3; or    -   (2) an amino acid sequence derived from the amino acid sequence        according to (1) above by substitution, deletion, and/or        addition of one or more amino acids, which specifically        recognizes sphingomyelin and has low cytotoxicity.

More specifically, the first protein of the present invention has eitherof the following an amino acid sequences:

-   -   (1) the amino acid sequence of SEQ ID NO: 3; or    -   (2) an amino acid sequence derived from the amino acid sequence        of SEQ ID NO: 3 by substitution, deletion, and/or addition of        one or more amino acids, which specifically recognizes        sphingomyelin and has low cytotoxicity.

The second protein of the present invention is a protein obtained bydeleting N terminal and/or C terminal of Eisenia foetida toxins Lysenin1 or 3, which specifically recognizes sphingomyelin. The portion to bedeleted in the amino acid sequence of Lysenin 1 or 3 may be N terminalor C terminal, and both of N terminal or C terminal may be deleted.Preferably, N terminal may be deleted. The number of the amino acidresidues to be deleted is not particularly limited so long as theobtained protein can specifically recognizes sphingomyelin. Preferably,at least 50 amino acid residues are deleted, and more preferably 70amino acid residues or more, and still more preferably 100 amino acidresidues can be deleted. The upper limit of the number of the amino acidresidues to be deleted is preferably 200 or less.

More specifically, the second protein of the present invention haseither of the following an amino acid sequences:

-   -   (1) the amino acid sequence of SEQ ID NO: 5; or    -   (2) an amino acid sequence derived from the amino acid sequence        of SEQ ID NO: 5 by substitution, deletion, and/or addition of        one or more amino acids, which specifically recognizes        sphingomyelin and has low cytotoxicity.

The range of “one or more” in the phrase “an amino acid sequence derivedfrom the amino acid sequence . . . by substitution, deletion, and/oraddition of one or more amino acids” is not particularly limited. Forexample, it means about 1 to 50, preferably 1 to 20, more preferably 1to 10, more preferably 1 to 7, still more preferably 1 to 5, andparticularly preferably 1 to 3.

The phrase “specifically recognizes sphingomyelin” used herein meansthat, for example, in ELISA (enzyme-linked immunosorbent assay), areaction with sphingomyelin is observed while reactivity withphosphatidylcholine is low.

In order for the protein of the present invention to specificallyrecognize sphingomyelin, among the amino acid residues, tryptophan (Trp)is preferably maintained. That is, in a preferred embodiment of thepresent invention, the 20th, the 116th, the 187th, the 245th, and the291st tryptophan residues in the amino acid sequence of SEQ ID NO: 3 inthe Sequence Listing are maintained, and the 70th, the 128th, and the174st tryptophan residues in the amino acid sequence of SEQ ID NO: 5 inthe Sequence Listing are maintained.

The term “cytotoxicity” used herein can be confirmed by measuring thehemolysis activity of the red blood cell. For example, the hemolysisactivity can be measured by adding test proteins at variousconcentrations to the sheep red blood cell, performing a reaction at 37°C. for 30 minutes, precipitating the blood cells by centrifugation, andthen measuring the amount of hemoglobin released by cell disruption asthe absorbancy at 405 nm. One feature of the protein of the presentinvention is that it has low cytotoxicity which is measured in a manneras described above.

Methods for obtaining and producing the protein of the present inventionare not particularly limited. The protein may be either of a chemicallysynthesized protein or a recombinant protein produced by generecombination techniques. A recombinant protein is preferred from theviewpoint of the large scale production with a relatively simpleprocedure.

When obtaining chemically synthesized proteins, for example, the proteinof the present invention can be synthesized according to chemicalsynthesis methods such as Fmoc synthesis (fluorenylmethyloxycarbonylsynthesis) and t-Boc synthesis (t-butyloxycarbonyl synthesis). Variouscommercially available peptide synthesizers can be used to synthesizethe protein of the present invention, for example, those manufactured bySowa Trading Co., Inc. (manufactured by Advanced Chem Tech, USA),Perkin-Elmer Japan (manufactured by Perkin-Elmer, USA), PharmaciaBiotech (manufactured by Pharmacia Biotech, Sweden), Aloka Co.(manufactured by Protein Technology Instrument, USA), KURABO INDUSTRIESLTD. (manufactured by Synthecell-Vega, USA), Nihon PerSeptive Limited(manufactured by PerSeptive, USA), and Shimadzu Corp.

The protein of the present invention can be produced as a recombinantprotein by obtaining DNA having a nucleotide sequence which encodes theprotein (for example, a nucleotide sequence of SEQ ID NO: 3 or SEQ IDNO: 4) or a variant or homologue thereof and introducing it into apreferable expression system. Production of an expression vector and atransformant, and production of a recombinant protein using it ishereinafter described.

Regarding the protein comprising an amino acid sequence derived from theamino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5 by substitution,deletion, and/or addition of one or more amino acids, which specificallyrecognizes sphingomyelin and has low cytotoxicity, an ordinarily skilledperson in the art can appropriately produce such protein based on theinformation on the nucleotide sequence of SEQ ID NO: 4 that shows anexample of a DNA sequence coding for the amino acid sequence of SEQ IDNO: 3, or the information on the nucleotide sequence of SEQ ID NO: 6that shows an example of a DNA sequence coding for the amino acidsequence of SEQ ID NO: 5.

(B) Genes of the Present Invention

The present invention also relates to a gene which encodes the proteinof the present invention.

Specific examples of the gene which encodes the first protein of presentinvention include genes having either of the following nucleotidesequences:

-   -   (1) the nucleotide sequence of SEQ ID NO: 4; or    -   (2) a nucleotide sequence derived from the nucleotide sequence        of SEQ ID NO: 4 by substitution, deletion, and/or addition of        one or more nucleotides, which specifically recognizes        sphingomyelin and codes for a protein having low cytotoxicity.

Specific examples of the gene which encodes the second protein ofpresent invention include genes having either of the followingnucleotide sequences:

-   -   (1) the nucleotide sequence of SEQ ID NO: 6; or    -   (2) a nucleotide sequence derived from the nucleotide sequence        of SEQ ID NO: 6 by substitution, deletion, and/or addition of        one or more nucleotides, which specifically recognizes        sphingomyelin and codes for a protein having low cytotoxicity.

The range of “one or more” in “a nucleotide sequence derived from thenucleotide sequence of SEQ ID NO: 4 or 6 by substitution, deletion,and/or addition of one or more nucleotides” is not particularly limited.For example, it means about 1 to 150, preferably 1 to 60, morepreferably 1 to 30, more preferably 1 to 20, still more preferably 1 to10, and particularly preferably 1 to 5.

A method for obtaining the gene of the present invention is notparticularly limited. Adequate primers can be prepared in accordancewith information on the nucleotide sequence and the amino acid sequenceaccording to SEQ ID NOs: 1 and 13 (the nucleotide sequence and the aminoacid sequence of Lysenin 1), SEQ ID NOs: 2 and 14 (the nucleotidesequence and the amino acid sequence of Lysenin 3), SEQ ID NO: 3 (theamino acid sequence of the chimera protein of the present inventioncomprising Lysenins 1 and 3 prepared in the following example 1), SEQ IDNO: 4 (the nucleotide sequence of the chimera protein of the presentinvention comprising Lysenins 1 and 3 prepared in the following example1), SEQ ID NO: 5 (the 118st to 297st amino acid residues of Lysenin 1),and SEQ ID NO: 6 (the nucleotide sequence encoding the 118st to 297stamino acid residues of Lysenin 1) in Sequence Listing provided herein.The gene of the present invention can be prepared by using such primers.

The gene (variant gene) comprising a “nucleotide sequence derived fromthe nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6 by substitution,deletion, and/or addition of one or more nucleotides, which encodes aprotein which specifically recognizes sphingomyelin and has lowcytotoxicity” can be prepared by any conventional methods for anordinarily skilled person in the art, such as chemical synthesis,genetic engineering or mutagenesis.

This type of variant gene can be obtained by, for example, utilizing DNAcomprising the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6, andintroducing variation into the DNA. More specifically, DNA having thenucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6 can be contactedwith an agent for mutagen, or can be subjected to ultravioletirradiation or a genetic engineering technique. Site-directedmutagenesis which is one genetic engineering technique is useful sincespecific mutation can be introduced into a specific position.Site-directed mutagenesis can be carried out in accordance with methodsdescribed in, for example, Molecular Cloning: A Laboratory Manual,2^(nd) Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989; or Current Protocols in Molecular Biology, Supplement 1-38, JohnWiley & Sons (1987-1997).

(C) Vector Having the Gene of the Present Invention

The gene of the present invention can be incorporated into a suitablevector, and can be used as a recombinant vector. A vector may beexpression vector or non-expression vector, and can be selecteddepending on its use.

As cloning vectors, those capable of autonomous replication inEscherichia coli K12 strain are preferred. Any vectors, such as a phagevector or plasmid vector, can be used. Specific examples thereof includeZAP Express, pBluescript II SK(+), Lambda ZAP II (manufactured byStratagene), λgt10, λgt11, and λTripIEx (manufactured by CloneTech),λExCell (manufactured by Pharmacia), pT7T318U (manufactured byPharmacia), pcD2, pMW218 (manufactured by Wako Pure Chemical Industries,Ltd.), pUC118 (manufactured by TAKARA SHUZO CO., LTD), and pQE-30(manufactured by QIAGEN).

A preferred expression vector used herein is one that is capable ofautonomous replication in a host cell, or one that is capable ofincorporation into a chromosome of a host cell. A vector containing apromoter in a such position that the gene of the present invention canbe expressed is used as an expression vector.

When a bacterium is used as the host cell, the expression vector forexpressing the gene of the present invention is preferably capable ofautonomous replication in the bacterium, and is also preferably arecombinant vector composed of a promoter, a ribosome binding sequence,the above-described DNA, and a transcription termination sequence. Agene controlling the promoter may be contained therein.

Expression vectors for bacteria include, for example, pBTrP2, pBTac1,and pBTac2 (commercially available from Boehringer Mannheim), pKK233-2(manufactured by Pharmacia), pSE280 (manufactured by Invitrogen),pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN),pQE-30 (manufactured by QIAGEN), pKYP10 (Japanese Patent ApplicationLaying-Open No. 58-110600), pKYP200 [Agrc. Biol. Chem., 48, 669 (1984)],PLSA1 [Agrc. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci.USA, 82, 4306 (1985)], pBluescrlptII SK+, pBluescriptII SK(−)(manufactured by Stratagene), pTrS30 (FERMBP-5407), pTrS32 (FERMBP-5408), pGEX (manufactured by Pharmacia), pET-3 (manufactured byNovagen), pTerm2 (U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094, U.S.Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC194, pUC18 [Gene, 33, 103(1985)], pUC19 [Gene, 33, 103 (1985)], pSTV28 (manufactured by TAKARASHUZO CO., LTD), pSTV29 (manufactured by TAKARA SHUZO CO., LTD), pUC118(manufactured by TAKARA SHUZO CO., LTD), and pQE-30 (manufactured byQIAGEN). Promoters for bacteria include, for example, a promoter derivedfrom Escherichia coli or phage such as a trp promoter (P trp), a lacpromoter (P lac), a P_(L) promoter, a P_(R) promoter, or a P_(SE)promoter; a SP01 promoter; a SP02 promoter, and a penP promoter.

Examples of expression vectors for yeast include YEp13 (ATCC37115),YEp24 (ATCC37051), Ycp5O (ATCC37419), pHS19, and pHS15. Promoters foryeast include, for example, a PHO5 promoter, a PGK promoter, a GAPpromoter, an ADH promoter, a gall promoter, a gal10 promoter, aheat-shock protein promoter, an MFα1 promoter, and a CUP1 promoter.

Examples of expression vectors for animal cells include pcDNAI, pcDM8(commercially available from Funakoshi), pAGE107 [Japanese PatentApplication Laying-Open No. 3-22979; Cytotechnology, 3, 133, (1990)],pAS3-3 (Japanese Patent Application Laying-Open No. 2-227075), pCDM8[Nature, 329, 840, (1987)], pcDNAI/AmP (manufactured by Invitrogen),pREP4 (manufactured by Invitrogen), pAGE103 [J. Blochem., 101, 1307(1987)], and pAGE210. Promoters for animal cells include, for example, apromoter of cytomegalovirus (human CMV) IE (immediate early) gene, anSV-40 early promoter, a retrovirus promoter, a metallothionein promoter,a heat-shock promoter, and an SRα promoter.

Examples of expression vectors for plant cells include plG121-Hm [PlantCell Report, 15, 809-814 (1995)] and pBI121 [EMBO J. 6, 3901-3907(1987)]. Promoters for plant cells include, for example, a cauliflowermosaic virus 35S promoter [Mol. Gen. Genet (1990) 220, 389-392] and aribulose bisphosphate carboxylase small subunit promoter.

(D) Transformant Having the Gene of the Present Invention

The transformant having the gene coding for the protein of the presentinvention can be produced by introducing the recombinant vector(preferably an expression vector) into a host.

Examples of bacteria host cells include microorganisms belonging to thegenus Escherichia, the genus Corynebacterium, the genus Brevibacterium,the genus Bacillus, the genus Microbacterium, the genus Serratia, thegenus Pseudomonas, the genus Agrobacterium, the genus Alicyclobacillus,the genus Anabaena, the genus Anacystis, the genus Arthrobacter, thegenus Azobacter, the genus Chromatium, the genus Erwinia, the genusMethylobacterium, the genus Phormidium, the genus Rhodobacter, the genusRhodopseudomonas, the genus Rhodospiri11um, the genus Scenedesmun, thegenus Streptomyces, the genus Synnecoccus, and the genus Zymomonas.Methods for introducing a recombinant vector into a bacteria host cellinclude, for example, the calcium ion method and the protoplast method.

Examples of a yeast host include Saccharomyces cerevisae,Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pu11ulans,and Schwanniomyces a11uvius. Methods for introducing a recombinantvector into a yeast host include, for example, an electroporationmethod, a spheroplast method, and a lithium acetate method.

Examples of animal host cells include Namalwa cell, COS1 cell, COS7cell, and CHO cell. Methods for introducing a recombinant vector intoanimal cells include, for example, an electroporation method, a calciumphosphate method, and a ripofection method.

When insect cells are used as the host, a recombinant gene introducingvector and a baculovirus are co-introduced into the insect cell, arecombinant virus is obtained in a supernatant of the insect cellculture, and the insect cell is infected with the recombinant virus.Thus, a protein can be expressed (for example, as described inBaculovirus Expression Vectors, A Laboratory Manual; and CurrentProtocols in Molecular Biology, Bio/Technology, 6, 47 (1988)).

Baculoviruses used herein include, for example, Autographa californicanuclear polyhedrosis virus, which is a virus infected with an insect ofspecies Mamestra.

Examples of insect cells used herein include Sf9 and Sf21, which areovarian cells of Spodoptera frugiperda [Baculovirus Expression Vectors,A Laboratory Manual, W. H. Freeman and Company (New York, 1992)], andHiFive, which is an ovarian cell of Trichoplusia ni (manufactured byInvitrogen).

Methods for co-introducing the recombinant gene introducing vector andthe baculovirus into the insect cell for the preparation of arecombinant virus include, for example, a calcium phosphate method and aripofection method.

(E) Production of a Recombinant Protein Using the Transformant of thePresent Invention

According to the present invention, a recombinant protein can beisolated by culturing a transformant having the gene of the presentinvention that is produced as above, producing and accumulating theprotein of the present invention in the cultured product, and collectingthe protein of the present invention from the cultured product.

When the transformant of the present invention is a prokaryotic organismsuch as Escherichia coli or an eukaryotic organism such as a yeast, themedium for culturing these microorganisms may be either a natural orsynthetic culture medium, as long as the medium comprises a carbonsource, a nitrogen source, an inorganic base and the like, which themicroorganism can assimilate, and the culturing of the transformant canbe efficiently carried out. The culturing is preferably carried outunder an aerobic condition, the culture temperature is generally from 15to 40° C., and the culture time is generally from 16 hours to 7 days.The pH value of the culture solution can be adjusted using an inorganicor organic acid, an alkaline solution, urea, calcium carbonate, ammoniaand the like. If necessary, antibiotics such as Ampicillin ortetracycline may be added to the medium while culturing.

As a medium for culturing the transformant prepared using animal cellsas the host cell, commonly used RPM11640 medium, Eagle's MEM medium orDMEM medium, or a medium prepared by adding fetal bovine serum or thelike to these mediums, are used. The culturing is generally carried out,for example, at a pH value of 6 to 8 at 30 to 40° C. in the presence of5% CO₂ for 1 to 7 days. While culturing, antibiotics such as kanamycinand penicillin may be added to the medium if necessary.

As a medium for culturing the transformant prepared using plant cells asthe host cell, commonly used media such as MS medium and R2P medium areused depending on the type of the plant. The culturing is generallycarried out at a pH value of 6 to 8 at 15 to 35° C. for 1 to 21 days.While culturing, antibiotics such as kanamycin and hygromycin may beadded to the medium if necessary.

In order to isolate and purify the protein of the present invention froma cultured product of the transformant, a conventionalisolation-purification method for the protein may be employed.

For example, when the protein of the present invention is expressed in adissolved state in the cell, after the completion of culturing, cellsare collected by centrifugation, suspended in an aqueous buffersolution, and fragmented using, for example, an ultrasonic crusher,French press, Menton-Gaulin homogenizer, or DYNO-Mill to obtain acell-free extract. The cell-free extract is centrifuged to obtain asupernatant. The resultant supernatant is subjected to conventionaltechniques for isolating and purifying proteins, that is, techniquessuch as solvent extraction, salting-out using ammonium sulfate and thelike, desalination, precipitation using organic solvents, anion exchangechromatography using resins such as diethyl amino ethyl (DEAE)sepharose, cation exchange chromatography using resins such asS-Sepharose FF (manufactured by Pharmacia), hydrophobic chromatographyusing resins such as butyl sepharose and phenyl sepharose, gelfiltration using a molecular sieve, affinity chromatography,chromatofocusing, and electrophoresis such as isoelectric focusing.These methods may be carried out alone or in combination of two or moreto obtain purified preparations.

(F) Reagent for Detecting Sphingomyelin

The present invention also relates to a reagent for detectingsphingomyelin which comprising the protein of the present invention. Theprotein of the present invention can specifically recognizesphingomyelin, and thus is useful as a reagent for detectingsphingomyelin. When the protein of the present invention is used as areagent for detecting sphingomyelin, it may be used alone or may be usedas a labeled protein that is labeled with substances for detection. Thetypes of labeling substances for detection are not particularly limitedand include, for example, affinity substances such as maltose-bindingprotein, biotin, avidin, and streptavidin; enzymes such as horseradishperoxidase and alkaline phosphatase; fluorescent substances such as Cy5and Cy3 (Amersham), fluorescein, tetramethylrhodamine, Texas Red, andacridine orange; chemiluminescent substances such as luminol andacridinium-1; and radioactive substances.

(G) Method for Detecting Sphingomyelin Using the Protein of the PresentInvention

The protein of the present invention specifically recognizessphingomyelin and has low cytotoxicity. Therefore, sphingomyelin can bedetected by using the protein of the present invention as a probe.

A method for detecting sphingomyelin is not particularly limited, andmay be an in vitro method such as ELISA or an in vivo method such ascell staining.

In order to perform ELISA, an ELISA plate is coated with asphingomyelin-containing sample and blocked with bovine serum albumin orthe like. Thereafter, the protein of the present invention is added tothe plate, and incubation is carried out. After washing the plate,suitable reagents for detecting the protein of the present invention aresuccessively added thereto and reacted, thereby detecting sphingomyelinin the sample. For example, when the protein of the present inventionhaving an MBP (maltose-binding protein) sequence is used as a detectingprobe, after the reaction with the sample, the sample is incubated withan anti-MBP rabbit serum, and is washed. Subsequently, incubation iscarried out using a biotin-labeled anti-rabbit antibody and horseradishperoxidase (HRP)-labeled streptavidin, color is developed witho-phenylendiamine, and the absorbancy at 492 nm is measured. Thus,sphingomyelin in the sample can be detected.

In order to perform cell staining, for example, after the cell isimmobilized on a cover slip, a plasma membrane is holed through adigitonin treatment, followed by reaction with the protein of thepresent invention. After the reaction, suitable reagents for detectingthe protein of the present invention are successively added thereto andreacted, thereby detecting sphingomyelin in the cell. For example, whenthe protein of the present invention having an MBP (maltose-bindingprotein) sequence is used as a detecting probe, after the reaction withthe cell, incubation is carried out using diluted anti-MBP rabbit serum(NEB) and a fluorescent-labeled anti-rabbit antibody, followed byinclusion. Then, cells are observed under a confocal microscope, therebydetecting sphingomyelin in the cell.

(H) Kit for Detecting Sphingomyelin

The protein, the gene, the vector, the transformant, and the reagent fordetecting sphingomyelin according to the present invention as describedherein, can be provided in the form of a kit for detectingsphingomyelin. The kit for detecting sphingomyelin can comprise theprotein, the gene, the vector, the transformant, or the reagent fordetecting sphingomyelin, as well as other necessary reagents if desired.Such other reagents include, for example, a buffer solution required inthe reaction, a blocking solution, washing solution, and other reagentsrequired in the detection of labels. A ordinarily skilled person in theart can appropriately select such reagents, and can construct a kit.

The present invention will now be described in more detail withreference to the following examples although it is not limited to theseexamples only.

EXAMPLES Example 1

(A) Cloning of cDNA of Earthworm Toxin Lysenin

Cloning of cDNA of earthworm toxin Lysenin has been already reported(Sekizawa, Y., Kubo, T., Kobayashi, H., Nakajima, T., and Natori, S.(1997), Molecular cloning of cDNA for lysenin, a novel protein in theearthworm Eisenia foetida that causes contraction of rat vascular smoothmuscle. Gene 191, 97-102). Also, the nucleotide sequences of Lysenin 1and Lysenin 3 are registered in a database (Lysenin 1: GenBank85846; andLysenin 3: GenBank85848). The nucleotide sequence and the amino acidsequence of Lysenin 1 are shown in SEQ ID NOs: 1 and 13 in SequenceListing, and the nucleotide sequence and the amino acid sequence ofLysenin 3 are shown in SEQ ID NOs: 2 and 14 in Sequence Listing.

(B) Production of MBP-Lysenin 1-3

(1) Production of pMBP-Lysenin 1, pMBP-Lysenin 3

In the amplification by PCR, the following primers in which the5′-terminus is BamHI site and the 3′-terminus is HindIII site wereprepared for both Lysenins 1 and 3. Lysenin 1 forward:ttcggatccatgtcggctaaagcagca (SEQ ID NO: 7) Lysenin 1 reverse:aagcttccgctttagttgcacctcatc (SEQ ID NO: 8) Lysenin 3 forward:ttcggatccatgtcgtctagagcagga (SEQ ID NO: 9) Lysenin 3 reverse:aagcttaaaacatgcggaagcaaatgt (SEQ ID NO: 10)

The full lengths of Lysenin 1 and Lysenin 3 were amplified by PCR usingcDNA obtained in (A) above as a template, and then were cloned intoPCR-TOPOII vector (Invitrogen). Thereafter, the complete nucleotidesequences were confirmed. The above plasmids were treated with BamHI andHindIII, a fragment containing Lysenin was collected and was ligated topMAL-C2X (New England Biolab) which was also treated with BamHI andHindIII, to obtain plasmids having an MBP (maltose-binding protein)sequence at the 5′ terminus, i.e., pMBP-Lysenin 1 and pMBP-Lysenin 3.

(2) Production of pMBP-Lysenin 1-3

EcoRI site is present at 141 bp in pMBP-Lysenin 1 and at 147 bp inpMBP-Lysenin 3. Also, pMAL-C2X has EcoRI site at a position of 2695 bp(BamHI is located at 2701 bp and HindIII at 2727 bp). pMBP-Lysenin 1 andpMBP-Lysenin 3 were respectively treated with EcoRI, and the 5′-terminusof Lysenin 3 was replaced with Lysenin 1. EcoRI corresponds to valine atthe 47th position, asparagin at the 48th position, and serine at the49th position in Lysenin 1, and EcoRI corresponds to leucine at the 49thposition, asparagin at the 50th position, and serine at the 51stposition in Lysenin 3. Therefore, Lysenin 1-3 is a chimera in which theregion from methionine at the 1st position to asparagin at the 48thposition is obtained from Lysenin 1, and the region from serine at the51st position to proline at the 300th position is obtained from Lysenin3.

(C) Expression of Protein

The above plasmid was transformed into Escherichia coli JM109 and, inaccordance with a conventional method, protein expression was inducedusing IPTG, bacteria were collected, and solubilization by sonicationwas carried out. Further, after affinity purification was carried outusing amylose resin (NEB), expression was confirmed by silver stainingand Western blotting using an anti-MBP antibody.

(D) Confirmation of Specific Reaction Between the Protein of the PresentInvention and Sphingomyelin by ELISA (Enzyme-Linked Immunosorbent Assay)

A 96-well ELISA plate (Immulon 1, Dynatech Laboratories) was coated with10 μM of sphingomyelin (SM) (phosphatidylcholine as control). Afterblocking with 3% bovine serum albumin (BSA), MBP-Lysenin 1, MBP-Lysenin3, and MBP-Lysenin 1-3 were respectively diluted into a suitable amount,and were added. The plate was incubated at room temperature for 2 hours.After the plate was washed three times, an anti-MBP rabbit serum (NEB)which was diluted to 1:1000, was added, and incubation was carried outfor 2 hours and the plate was then washed. Further, incubation wascarried out using a biotin-labeled anti-rabbit antibody and horseradishperoxidase (HRP)-labeled streptavidin, color was developed witho-phenylendiamine, and the absorbancy at 492 nm (630 nm as control) wasmeasured.

The results are shown in FIG. 1, left graph, and in FIG. 3. Theseresults indicate that MBP-Lysenin 1-3 specifically binds tosphingomyelin, as is the case with MBP-Lysenin 1 and MBP-Lysenin 3.

(E) Hemolysis

MBP-Lysenin 1, MBP-Lysenin 3 and MBP-Lysenin 1-3 at variousconcentrations were added to 3×10⁷ cells/mL of the sheep red blood cell,followed by a reaction at 37° C. for 30 minutes. The blood cells wereprecipitated by centrifugation, and the amount of hemoglobin released bycell disruption was measured as the absorbancy at 405 nm. The valueobtained when all the cells were disrupted was taken as 100%, and valuesat each point were calculated.

The results are shown in FIG. 1, right graph. These results indicatethat MBP-Lysenin 1 and MBP-Lysenin 3 exhibit hemolysis activity at acertain concentration or higher, while MBP-Lysenin 1-3 does not exhibithemolysis activity even at a high concentration.

(F) Cell Staining Using Niemann-Pick Type A Fibroblast

A fibroblast obtained from a patient suffering from Niemann-Pick diseasetype A was cultured on a glass cover slip for several days. After fixingwith paraformaldehyde, a plasma membrane was holed by a digitonintreatment, followed by reaction with MBP-Lysenin 1, MBP-Lysenin-3, orMBP-Lysenin 1-3. Incubation was carried out using diluted anti-MBPrabbit serum (NEB) and a fluorescent-labeled anti-rabbit antibody,followed by inclusion, and observation under a confocal microscope.

The results are shown in FIG. 2. The results shown in FIG. 2 indicatethat sphingomyelin of the fibroblast can be detected even in vivo byusing MBP-Lysenin 1-3 of the present invention

Example 2

(A) Cloning of cDNA of Earthworm Toxin Lysenin

Cloning of cDNA of earthworm toxin Lysenin has been already reported(Sekizawa, Y., Kubo, T., Kobayashi, H., Nakajima, T., and Natori, S.(1997), Molecular cloning of cDNA for lysenin, a novel protein in theearthworm Eisenia foetida that causes contraction of rat vascular smoothmuscle. Gene 191, 97-102). Also, the nucleotide sequence of Lysenin 1 isregistered in a database (GenBank85846). The nucleotide sequence and theamino acid sequence of Lysenin 1 are shown in SEQ ID NO: 1 in SequenceListing.

(B) Method for Producing MBP-Lysenin 118-297

In amplification by PCR, the following primers capable of amplifying afragment which is equivalent to amino acids 118-297 in Lysenin 1(containing nucleotides 351-894 in the nucleotide sequence) and hasBamHI site at the 5′-terminus and HindIII site at the 3′-terminus, wereprepared. Lysenin 118 forward: ttcggatccctgaatgxtgacgttggtgga (SEQ IDNO: 11) Lysenin 1 reverse: aagcttccgctttagttgcacctcatc (SEQ ID NO: 12)

The amino acids 118-297 in Lysenin 1 were amplified by PCR using theabove primers using cDNA of Lysenin 1 obtained in (A) above as atemplate, and then cloned into PCR-TOPOII vector (Invitrogen).Thereafter, a complete nucleotide sequence was confirmed. The aboveplasmid was treated with BamHI and HindIII, a fragment containingLysenin was collected and was ligated to pMAL-C2X (New England Biolab)which was also treated with BamHI and HindIII, to obtain a plasmidhaving an MBP (maltose-binding protein) sequence at its 5′ terminus,i.e., pMBP-Lysenin 118-297.

(C) Expression of Protein

The plasmid obtained in above (B) was transformed into Escherichia coliJM109 and, in accordance with a conventional method, protein expressionwas induced using IPTG, bacteria were collected, and solubilization bysonication was carried out. Further, after affinity purification wascarried out using amylose resin (NEB), expression was confirmed bysilver staining and Western blotting using an anti-MBP antibody.

(D) Confirmation of Specific Reactivity Between the Protein of thePresent Invention and Sphingomyelin by ELISA (Enzyme-LinkedImmunosorbent Assay)

ELISA (enzyme-linked immunosorbent assay) was performed as follows.

A 96-well ELISA plate (Immulon 1, Dynatech Laboratories) was coated with10 μM of sphingomyelin (phosphatidylcholine as control). After blockingwith 3% bovine serum albumin (BSA), MBP-Lysenin 1, MBP, and MBP-Lysenin118-297 were respectively diluted into a suitable amount, and wereadded. The plate was incubated at room temperature for 2 hours. Afterthe plate was washed three times, an anti-MBP rabbit serum (NEB) whichwas diluted to 1:1000, was added, and incubation was carried out for 2hours and the plate was then washed. Further, incubation was carried outusing a biotin-labeled anti-rabbit antibody and horseradish peroxidase(HRP)-labeled streptavidin, color was developed with o-phenylendiamine,and the absorbancy at 492 nm (630 nm as control) was measured.

The results are shown in FIG. 4. The results of FIG. 4 indicate thatMBP-Lysenin 118-297 specifically binds to sphingomyelin (SM), as is thecase with MBP-Lysenin 1.

(E) Hemolysis

MBP-Lysenin 1 and MBP-Lysenin 118-297 at various concentrations wereadded to 3×10⁷ cells/mL of the sheep red blood cell, followed by areaction at 37° C. for 30 minutes. The blood cells were precipitated bycentrifugation, and the amount of hemoglobin released by cell disruptionwas measured as the absorbancy at 405 nm. The value obtained when allthe cells were disrupted was taken as 100%, and values at each pointwere calculated.

The results are shown in FIG. 5. The results of FIG. 5 indicate thatthat MBP-Lysenin 1 exhibits hemolysis activity at a certainconcentration or higher, while MBP-Lysenin 118-297 (indicated as “del118-297” in FIG. 5) does not exhibit hemolysis activity even at a highconcentration.

(F) Cell Staining Using Niemann-Pick Type a Fibroblast

A fibroblast obtained from a patient suffering from Niemann-Pick diseasetype A was cultured on a glass cover slip for several days. After fixingwith paraformaldehyde, a plasma membrane was holed by a digitonintreatment, followed by reaction with MBP-Lysenin 118-297, MBP-Lysenin 1,or MBP. Incubation was carried out using diluted anti-MBP rabbit serum(NEB) and a fluorescent-labeled anti-rabbit antibody, followed byinclusion, and observation under a confocal microscope. At the sametime, incubation was carried out using a monoclonal antibody (CALTAG)obtained from a mouse against protein CD 63 existing in the lateendosome and a fluorescent-labeled anti-mouse antibody, thereby the lateendosome was visualized.

The results are shown in FIG. 6. The results shown in FIG. 2 indicatethat sphingomyelin of the fibroblast can be detected even in vivo byusing MBP-Lysenin 118-297 of the present invention

A Niemann-Pick type A fibroblast was stained in the same manner asdescribed above. The obtained results are shown in FIG. 7. The resultsindicate that MBP-61-297, MBP-131-297, MBP-146-297, and MBP-161-297 canbe detected in vivo, and suggest that MBP-61-246, MBP-61-207, andMBP-208-297 do not recognize sphingomyelin.

INDUSTRIAL APPLICABILITY

According to the present invention, there can be provided a protein thatis usable as a sphingomyelin detecting probe which specificallyrecognizes sphingomyelin and has low cytotoxicity.

1. A protein which is obtained by deleting N terminal and/or C terminalfrom earthworm toxins Lysenin 1 or 3, and which specifically recognizessphingomyelin.
 2. The protein of claim 1 which is obtained by deleting50 to 200 amino acid residues of N terminal and/or C terminal ofearthworm toxins Lysenin 1 or
 3. 3. A protein having either of thefollowing amino acid sequences: (1) the amino acid sequence of SEQ IDNO: 5; or (2) an isolated amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 5 by substitution, deletion, and/or additionof one or more amino acids, which specifically recognizes sphingomyelinand has low cytotoxicity.
 4. A gene which encodes the protein of claim3.
 5. A gene having either of the following nucleotide sequences: (1)the nucleotide sequence of SEQ ID NO: 6; or (2) an isolated nucleotidesequence derived from the nucleotide sequence of SEQ ID NO: 6 bysubstitution, deletion, and/or addition of one or more nucleotides,which encodes a protein which specifically recognizes sphingomyelin andhas low cytotoxicity.
 6. A vector having the gene of claim
 4. 7. Atransformant having the vector of claim
 6. 8. A reagent composition fordetecting sphingomyelin comprising the protein of claim
 1. 9. A methodfor detecting sphingomyelin comprising: (1) adding the protein of claim1 as a probe to a sample; (2) adding at least one reagent; and (3)detecting sphingomyelin in the sample.
 10. A kit for detectingsphingomyelin, which comprises: (a) a protein which is obtained bydeleting N terminal and/or C terminal from earthworm toxins Lysenin 1 or3, and which specifically recognizes sphingomyelin, or (b) a proteinwhich is obtained by deleting N terminal and/or C terminal fromearthworm toxins Lysenin 1 or 3, and which specifically recognizessphingomyelin and which is obtained by deleting 50 to 200 amino acidresidues of N terminal and/or C terminal of earthworm toxins Lysenin 1or 3, or (c) a protein having either of the following amino acidsequences: (1) amino acid sequence of SEQ ID NO: 5; or (2) an amino acidsequence derived from the amino acid sequence of SEQ ID NO: 5 bysubstitution, deletion, and/or addition of one or more amino acids whichspecifically recognizes sphingomyelin, and has low cytotoxicity, or (d)a gene which encodes the protein of (c) above, or (e) a gene havingeither of the following nucleotide sequences: (1) the nucleotidesequence of SEQ ID NO: 6; or (2) a nucleotide sequence derived from thenucleotide sequence of SEQ ID NO: 6 by substitution, deletion, and/oraddition of one or more nucleotides, which encodes a protein whichspecifically recognizes sphingomyelin, and has low cytotoxicity, or avector of the gene of (d), or a transformant of the vector of the geneof (d), or the reagent for detecting sphingomyelin comprising theprotein (a).